1. Field of Invention
The present invention is a method for converting recombinant procaspase 8 protein to active human caspase 8 enzyme and a method of producing the recombinant protein.
2. Related Art
The caspases are a family of related cysteinyl proteinases which play important intracellular roles in inflammation and apoptosis. See, for example, D. W. Nicholson et al., TIBS 22: 299-306 (1997). Members of this proteinase family share a number of features in common. They all employ a conserved cysteine residue as the nucleophile for attack on peptide bonds, and the sites of cleavage all show Asp in the P.sub.1 position of the peptide substrates (A. C. Chinnayan et al., Current Biol. 6, 555-562 (1996)). The catalytic domain of the caspases has a mass of roughly 30 kDa and comprises two polypeptide chains, a 20 kDa N-terminal a fragment which contains the active site Cys, and a 10 kDa C-terminal .beta. peptide which contributes to the formation of the active site. These chains arise by internal cleavage of a single-chain zymogen precursor, and are tightly associated in an .alpha..beta. heterodimer. Proteolytic processing which gives rise to these component polypeptides may be autocatalytic (H. R. Stennicke et al., J. Biol. Chem. 272, 25719-25723 (1997)), or can be mediated by other caspases or by enzymes of similar specificity, for example, granzyme B, a lymphocyte enzyme with a specificity for P.sub.1 Asp similar to that of the caspases (C. J. Froelich et al., Nature Medicine 3: 954-55 (1997)).
As with nearly all proteolytic enzymes, the caspases exist as inactive precursors, or proenzymes. The length of the N-terminal prodomains of the caspases vary considerably depending on how activation is regulated. Our present invention focuses on caspase 8 which is involved in apoptosis. The structure of the proenzyme, or zymogen form of this protein is illustrated schematically in FIG. 1. The N-terminal prosegment in caspase 8, contains two death-effector domains (DED). Immunoprecipitation experiments of activated death-initiation signaling complexes (DISC) show that procaspase 8 is a component of the activated receptor complexes (M. Muzio et al., J. BioL Chem. 273, 2926-2930 (1996)). Because of sequence homology between the death effector domains of procaspase 8 and the death domains (DD) of FADD and TRADD, the death domain proteins associated with TNF and Fas receptors (J. P. Medema et al., EMBO Journal 16, 2794-2804 (1997)), the regions of homology are thought to result in the selective association or recruitment of procaspase 8 to the activated receptors. As a component of activated DISC, procaspase 8 plays a key role in the direct line of signal transduction, and activation of the Fas or TNF receptors with Fas ligand or TNF results in its autocatalytic, intermolecular activation. Activated caspase 8 is then thought to activate other, downstream caspases, like caspase 3, which have prosegments that are shorter and whose intracellular concentrations are not high enough to support their autocatalytic processing and activation. Thus, caspase 8 has been suggested to sit at the apex of the Fas or TNF mediated apoptotic cascade and likely serves as the prime mover for activation of the "executioners" of apoptosis whose function is to destroy critical cellular proteins (D. W. Nicholson et al., see above).
Because of its initiating role in Fas or TNF mediated apoptosis, caspase 8 is believed to be a viable target in blockade of the undesirable cell death which occurs in a variety of neurological disorders. See, for example, M. P. Mattson et al., J. Neurochem. 70, 1-14 (1998)). In view of the biological significant role of caspase 8 in the apoptotic process and the need to further elucidate the role and mechanism of action of this protein in the cell death process the need for a reproducible method for the production of active protein has become very important.
The current belief that the death-effector domains provide the mechanism for recruitment of procaspase 8 to the cell membrane is consistent with our finding that given a sufficiently high concentration of the catalytic domain of the protein in solution, it is possible to affect autoactivation of a truncated procaspase 8 construct missing the DED.
Information Disclosure
References relating to caspase 8: M. Muzio et al., J. BioL Chem. 272, 2952-2956 (1997); H. R. Stennicke et al., J. Biol. Chem. 272, 25719-25723 (1997); Q. Zhou et al., J. Biol. Chem. 272, 7797-7800 (1997); D. A. Martin et al., J. Biol. Chem. 273, 4345-4349 (1998); and M. Muzio et al., J. Biol. Chem. 273, 2926-2930 (1998).
Other caspase references: J. Sun et al., Biochem. Biophys. Res. Comm. 238, 920-924 (1997); D. K Miller et al., J. Cell. Biochem. 64, 2-10 (1997); N. Margolin et al., J. Biol. Chem. 272, 7223-7228 (1997); R V. Talanian et al., J. Biol. Chem. 272, 9677-9682 (1997); N. A. Thornberry et al., J. Biol. Chem. 272, 17907-17911 (1997); K. 0111 et al, J. Biol. Chem. 271, 2097720980 (1996); and T. Fernandes-Alnemri et al., Cancer Res. 55, 2737-2742 (1995).